Intellectual Merit. Heme-iron containing peroxidases are enzymes that catalyze organic substrate oxidation while reducing hydrogen peroxide to water. Two novel heme peroxidases isolated from marine sea worms, one able to incorporate halide ions such as chloride and bromide into aromatic molecules and the other able to remove them, will be studied. The haloperoxidase, Notomastus lobatus chloroperoxidase (NCPO), halogenates phenols and is unusual in requiring an organic flavin component. Amphitrite ornata dehaloperoxidase (DHP) dehalogenates halophenols. Surprisingly, the structure of DHP is that of a globin - the same protein fold found in hemo- and myoglobin (Mb). It is the first known enzymatic globin. The unique structural properties of these two peroxidases bring into question whether they utilize a new peroxidase mechanism. A four-prong hypothesis-driven experimental plan will be followed. First, rapid kinetics experiments will probe the mechanism of DHP and NCPO catalysis and directly determine whether the traditional or an alternative mechanism is employed. Second, the ability of DHP and NCPO to carry out standard peroxidase reactions will be extensively tested in order to advance our understanding of DHP and NCPO function. Third, designed DHP and Mb mutants will be prepared to probe the mechanistic role of specific amino acids near the DHP heme and explore factors in the heme/globin environment that enhance DHP function relative to Mb. Fourth, NCPO will be cloned, expressed and structurally characterized. Small NCPO crystals have been obtained. NCPO is a complex multi-subunit hemoflavoenzyme; the roles of its subunits are unknown. Such enzymes with peroxidase activity have not yet been reported. Studies of its structure and mechanism will provide new information about halogenation in higher organisms. Investigations of complex multi-subunit-containing systems, such as those proposed herein, are at the cutting edge of mechanistic enzymology. Overall, the two novel peroxidases challenge the current view of how peroxidases function. The research will probe how nature has redesigned small globin-fold proteins into peroxidases and thereby will extend the boundaries of our understanding of structure/function relations in such enzymes.
Broader Impacts. The research combines the diverse disciplines of chemistry, molecular genetics and evolution, structural biology, and spectroscopy, and promotes interaction among chemistry and biology faculty and students via regular meetings of the three research groups. Students, from high school to post-graduate, will be trained. Undergraduates will continue to make significant research contributions. The three PIs have strong records training underrepresented minorities recently including two African-American students. One of the PIs directs SCienceLab, an outreach program for middle/high school science teachers and students. A specific 'Enzyme-Catalyzed Toxic Cleanup' SCiLab designed around biological dehaloperoxidases will provide a daylong hands-on, inquiry-based lab experience and convey the excitement of interfacing biological, chemical and environmental research. The goal is to show students how biological catalysts might be used in bioremediation technology. Moreover, the research results will continue to be used teaching graduate enzymology, crystallography and metallobiochemistry courses. The fundamental chemical/biochemical progress achieved may lead to the development of applications in the broader areas of biotechnology and bioremediation and will impact related ecological and environmental research.
This project has contributed to an improved understanding of how, in the process of molecular evolution, novel functions appear. Globins are among the oldest known family of proteins with a primary function of oxygen transport and storage. Dehaloperoxidase from the polychaete worm Amphitrite ornata is a bifunctional protein from the globin family that not only exhibits the oxygen binding capabilities (in the ferrous state) seen for other globins, but also has peroxidase catalytic properties (in the ferric state). Since oxygen binding by globins is universal and found in species older than polychaete worms, it is apparent that the peroxidase function evolved in this globin later. Bifunctional proteins are seldom observed; usually gene duplication and divergence takes place. Studies of the properties of this protein "in transition" have led to a better understanding of how new molecular properties emerge. The support from this award enabled five young scientists to receive their Ph.D. degrees in Chemistry from the University of South Carolina. We have determined three crystal structures of DHP mutants forming complexes with TCP. Two alternative binding modes were observed: one with a TCP molecule bound deep in the distal pocket, at the heme α edge, the other with a TCP molecule bound at the entrance to the distal cavity, at the heme β edge. Analysis of the protein – ligand interactions in these structures led us to conclude that both modes of binding are not productive but inhibitory because the hydrogen peroxide binding site is blocked. Consequently the reaction proceeds through the standard peroxidase mechanism: hydrogen peroxide binds first, generates active intermediates which bind and oxidase halophenols. This is contrary to the alternative order of substrates binding recently advanced by another laboratory. Moreover, we have proposed that the α-edge binding site is related to heme oxidation, and thus activation of the peroxidase activity, observed in the presence of TCP. The β-edge binding site appears to be related to the catalytic binding site and is analogous to that observed in the classical peroxidases. We have proposed a mechanism for DHP in which hydrogen peroxide binds first and is cleaved to form Compound I. The distal histidine moves out of the cavity and recruits TCP by forming a hydrogen bond to its hydroxyl, as observed in the DHP•TCP complex. The Cl-4 atom is in the vicinity of the active oxygen of Compound I and is the likely site of electron transfer. Mutational studies we have published are consistent with this hypothesis. Out kinetic data indicate that the binding of TCP to DHP in the ferrous state is much weaker than to Compound I. We have determined the apparent Km values of TCP, which reflect the affinity of DHP activated by peroxide; they are two orders of magnitude lower than the concentrations needed to observe TCP binding in crystal soaking experiments in the absence of peroxide. We have reached the same conclusions while studying the dehaloperoxidase activity of myoglobin. Thus kinetic data corroborate the classical peroxidase mechanism. The dehaloperoxidase activity of horseradish peroxidase Compounds I and II with halophenol substrates was examined under true single turnover conditions (i.e., no excess H2O2). This study provided further support for our now-general conclusion that the mechanism of halophenol dehalogenation by peroxidases involves two consecutive one-electron steps with a dissociable radical intermediate. Our discovery that DHP peroxidase function is activated by TCP entirely changed our perspective on DHP biology. It solved the main paradox: why did A. ornata evolve its globin to become a peroxidase rather than picking a gene for a classical peroxidase, which are in abundance. In retrospect, it is obvious. Peroxidases produce reactive oxygen species which are harmful. So the ideal halophenol detoxification mechanism would involve a low-activity, substrate activated enzyme which can easily be deactivated. And this description perfectly fits DHP. Two isoproteins, DHP A and DHP B, are present in A. ornata. They are coded by two genes, but are very closely related. The differences are limited to five amino acids. Thus they represent products of gene duplication and very limited divergence. Initially we had a simplistic view, two functions - two genes so the optimization of the new function (peroxidase) should take place. We expressed the mutants on the path from DHP A to DHP B and we are in the process of their characterization with regards to their three essential functions: 1) oxygen binding, 2) switching from ferrous to the ferric state, 3) dehaloperoxidase activity. Our data indicate that while DHP B has three times higher dehaloperoxidase activity, it has also somewhat higher oxygen binding affinity. The main difference is in the rate of switching from the ferrous to the ferric state and this confirms our thesis that the on/off switching is the crucial aspect of DHP biology.
